The human gut is the most densely inhabited ecosystem on Earth (Marchesi and Shanahan, 2007). Like other complex microbial ecosystems, the human microbiota has not been sampled to completion (Eckburg et al., 2005). This is because the individual species of the gut microbiota are difficult to culture axenically in vitro (Hart et al., 2002). In fact, of the 500+ bacterial species which colonize the human intestinal tract, about 75% have not been cultured using conventional techniques (Duncan et al., 2007; Eckburg et al., 2005; Hayashi et al., 2002). It is recognized that novel culture techniques are required to grow these “unculturable” microorganisms.
Studies of gut microbiota have been hampered by a lack of model systems. While in vivo models can provide researchers with physiologically relevant experimental models, they have several drawbacks. For example, different study participants can each have unique, host-specific community profiles representing their gut microbiota, making comparison of the gut microbiota between subjects difficult, especially when attempting to correlate the effects of a treatment to changes in the gut microbiota. In vivo models also often limit the dynamic monitoring of the gut microbiota by deriving their data from end-point measurements. Experiments involving humans or animals require research ethics approval which can limit the experiments conducted on an individual's gut microbiota in vivo.
In an attempt to improve upon the drawbacks of in vivo models, several in vitro models have been developed. These in vitro systems range from simple batch culture vessels to complex continuous culture or ‘chemostat’ systems (Macfarlane, G. T. and Macfarlane, S., Curr. Opin. Biotechnol., 18(2): 156-62, 2007). Using chemostats, communities seeded from fresh feces can reach a steady-state that closely resembles in vivo distal gut communities. Being a host-free system, chemostats supporting gut microbiota make ideal vessels in which to study microbial perturbations that directly result from the addition of exogenous stimuli in isolation from the effects of these stimuli on host physiology, making them useful for mechanistic studies (Macfarlane, G. T. and Macfarlane, S., Curr. Opin. Biotechnol., 18(2): 156-62, 2007).
In vitro models also provide several other advantages over in vivo models in studies of the human gut microbiota. In vitro studies are generally inexpensive and easy to set-up. They also allow for the strict control of factors that influence the environment while still facilitating frequent and simple sampling of the simulated gut communities. However, while chemostats provide a useful tool to investigate the microbial ecology of the gut, operational parameters vary widely between different models in different laboratories, often without experimental validation. Preparation of the inocula, composition of the media, and retention time of the vessel are parameters which can vary between different studies.
To represent a valid model of the human distal gut, communities which develop within chemostat vessels should share some similarity to the fecal inoculum from which the gut community was derived. The microbial ecosystem of the gut is a highly diverse community, and it is therefore important that communities grown in artificial systems also retain a high level of diversity (including species richness and evenness). Finally, the reproducibility and stability of these communities must be established and characterized before experimentation can begin. This means that microbial communities developed within these models must be thoroughly analyzed and compared to in vivo communities before the validity of a system can be confirmed.
Chemostat and fecal communities can be monitored using molecular methods such as Denaturing Gradient Gel Electrophoresis (DGGE). Currently, there is a lack of standardization between DGGE analysis methods used in different research laboratories. Methods of DGGE analysis vary from visual inspection to methods utilizing statistical analysis software (such as GelCompar™, BioNumerics, GeneTools, Quantity One™, etc.). Monitoring of communities using computer software allows for more reliable and detailed analysis of DGGE gels and provides more data on the composition and structure of microbial communities, the stability of the community, and the similarity between profiles. However, laboratories utilizing these analysis programs do not use consistent methods when analyzing their DGGE gels and report varying data on their communities. If the analysis of DGGE gels can be standardized then this will facilitate the comparison between the chemostat communities from different laboratories.
It would be desirable therefore to be provided with chemostat models of the human distal gut that are stable, reproducible and biologically significant, as well as more complete methods for the assessment and verification of such models.